Feasibility of the preparation of cochleate suspensions from naturally derived phosphatidylserines.

Introduction: Cochleates are cylindrical particles composed of dehydrated phospholipid bilayers. They are typically prepared by addition of calcium ions to vesicles composed of negatively charged phospholipids such as phosphatidylserines (PS). Due to their high physical and chemical stability, they provide an interesting alternative over other lipid-based drug formulations for example to improve oral bioavailability or to obtain a parenteral sustained-release formulation. Methods: In the present study, the feasibility to prepare cochleate suspensions from soy lecithin-derived phosphatidylserines (SPS) was investigated and compared to the "gold standard" dioleoyl-phosphatidylserine (DOPS) cochleates. The SPS lipids covered a large range of purities between 53 and >96% and computer-controlled mixing was evaluated for the preparation of the cochleate suspensions. Electron microscopic investigations were combined with small-angle x-ray diffraction (SAXD) and Laurdan generalized polarization (GP) analysis to characterize particle structure and lipid organization. Results: Despite some differences in particle morphology, cochleate suspensions with similar internal lipid structure as DOPS cochleates could be prepared from SPS with high headgroup purity (≥96%). Suspensions prepared from SPS with lower purity still revealed a remarkably high degree of lipid dehydration and well-organized lamellar structure. However, the particle shape was less defined, and the typical cochleate cylinders could only be detected in suspensions prepared with higher amount of calcium ions. Finally, the study proves the feasibility to prepare suspensions of cochleates or cochleate-like particles directly from a calcium salt of soy-PS by dialysis.

Drastic differences between the release kinetics of two highly related porphyrins in liposomal membranes: mTHPP and pTHPP.

HYPOTHESIS: The release of hydrophobic compounds from liposomal membranes occurs by partitioning and is thus determined by the physicochemical properties (e.g. logP and water solubility) of the drug. We postulate that even minor structural differences, e.g. the position of the phenolic OH-group of the hydrophobic porphyrins mTHPP and pTHPP (meta vs. para substitution), distinctly affect their partitioning and release behavior from liposomes. EXPERIMENTS: The release and redistribution of mTHPP and pTHPP from lecithin or POPC/POPG liposomes to different acceptor particles (DSPE-mPEG micelles and liposomes) was studied by asymmetrical flow field-flow fractionation to separate donor and acceptor particles. Reversed phase HPLC was applied to detect differences in partitioning. Molecular dynamics (MD) simulations were carried out to obtain molecular insight in the different behavior of the two compounds inside a lipid bilayer. FINDINGS: Despite the minor differences in chemical structure, mTHPP is more hydrophobic and redistributes much slower to both acceptor phases than pTHPP. MD simulations indicate that compared to pTHPP, mTHPP makes stronger hydrogen bonds with the lipid head groups, is oriented more parallel to the lipid tails and is embedded slightly deeper in the membrane.

The ROC skin model: A robust skin equivalent for permeation and live cell imaging studies.

Rat epidermal keratinocyte (REK) Organotypic culture (ROC) is an epidermis model that is robust and inexpensive to develop and maintain, and it has in previous studies been shown to have permeability characteristics close to those of human skin. Here, we characterize the model further by structural comparison to native human and rat skin and by investigating functional characteristics of lipid packing, polarity, and permeability coefficients. We show that the ROC model has structural similarities to native human skin and observe human skin-like permeability coefficients for testosterone and mannitol. We develop a transwell device that allows live cell microscopy of the tissue at the air-liquid interface and establish transgenic cell lines expressing different fluorescently tagged proteins. This enables showing the migration of keratinocytes during the first days after seeding, finding that keratinocytes have a higher mean migration rate in the earlier days of development. Collectively, our results show that the ROC model is an inexpensive and robust epidermis model that works reproducibly across laboratories.

Phosphatidylinositol Stabilizes Fluid-Phase Liposomes Loaded with a Melphalan Lipophilic Prodrug.

Previously, a liposomal formulation of a chemotherapeutic agent melphalan (Mlph) incorporated in a fluid lipid bilayer of natural phospholipids in the form of dioleoylglyceride ester (MlphDG) was developed and the antitumor effect was confirmed in mouse models. The formulation composed of egg phosphatidylcholine (ePC), soybean phosphatidylinositol (PI), and MlphDG (8:1:1, by mol) showed stability in human serum for at least 4-5 h. On the contrary, replacing PI with pegylation of the liposomes, promoted fast dissociation of the components from the bilayer. In this work, interactions of MlphDG-liposomes with the most abundant plasma protein-albumin-in function of the presence of PI in the formulation were explored using Fourier transform infrared spectroscopy. The release of MlphDG from the liposomes was studied by asymmetrical flow field-flow fractionation (AF4) using micelles formed by a polyethylene glycol conjugate with phosphatidylethanolamine to mimic the physiological lipid sink like lipoproteins. Our results show that PI actually protects the membrane of MlphDG-liposomes from the protein penetration, presumably due to pairing between the positively charged MlphDG and negatively charged PI, which compensates for the heterogeneity of the lipid bilayer. The AF4 technique also evidences high stability of the formulation as a drug carrier.

Bone Morphogenetic Protein 2 (BMP-2) Aggregates Can be Solubilized by Albumin-Investigation of BMP-2 Aggregation by Light Scattering and Electrophoresis.

Bone morphogenetic protein 2 (BMP-2) has a high tendency to aggregate at physiological pH and physiological ionic strength, which can complicate the development of growth factor delivery systems. The aggregation behavior in differently concentrated BMP-2 solutions was investigated using dynamic and static light scattering. It was found that at higher concentrations larger aggregates are formed, whose size decreases again with increasing dilution. A solubilizing effect and therefore less aggregation was observed upon the addition of albumin. Imaged capillary isoelectric focusing and the simulation of the surface charges of BMP-2 were used to find a possible explanation for the unusually low solubility of BMP-2 at physiological pH. In addition to hydrophobic interactions, attractive electrostatic interactions might be decisive in the aggregation of BMP-2 due to the particular distribution of surface charges. These results help to better understand the solubility behavior of BMP-2 and thus support future pharmaceutical research and the development of new strategies for the augmentation of bone healing.

In situ Gelling Amphotericin B Nanofibers: A New Option for the Treatment of Keratomycosis.

The purpose of our research was the development of Amphotericin B-loaded in situ gelling nanofibers for the treatment of keratomycosis. Different formulation strategies were applied to increase the drug load of the sparingly water-soluble Amphotericin B in electrospun Gellan Gum/Pullulan fibers. These include bile salt addition, encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and formation of a polymeric Amphotericin B polyelectrolyte complex. The Amphotericin B polyelectrolyte complex (AmpB-Eu L) performed best and was very effective against the fungal strain Issatchenkia orientalis in vitro. The complex was characterized in detail by attenuated total reflection infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry. A heat induced stress test was carried out to ensure the stability of the polyelectrolyte complex. To gain information about the cellular tolerance of the developed polyelectrolyte complex a new, innovative multilayered-stratified human cornea cell model was used for determination of the cellular toxicity in vitro. For a safe therapy, the applied ophthalmic drug delivery system has to be sterile. Sterilization by electron irradiation caused not degradation of pure Amphotericin B and also for the bile salt complex. Furthermore, the developed Amphotericin B polyelectrolyte complex was not degraded by the irradiation process. In conclusion, a new polyelectrolyte Amphotericin B complex has been found which retains the antifungal activity of the drug with sufficient stability against irradiation-sterilization induced drug degradation. Furthermore, in comparison with the conventional used eye drop formulation, the new AmpB-complex loaded nanofibers were less toxic to cornea cells in vitro. Electrospinning of the Amphotericin B polyelectrolyte complex with Gellan Gum/ Pullulan leads to the formation of nanofibers with in situ gelling properties, which is a new and promising option for the treatment of keratomycosis.

Control over Particle Size Distribution by Autoclaving Poloxamer-Stabilized Trimyristin Nanodispersions.

Lipid nanoparticles are under investigation as delivery systems for poorly water-soluble drugs. The particle size in these dispersions strongly influences important pharmaceutical properties like biodistribution and drug loading capacity; it should be below 500 nm for direct injection into the bloodstream. Consequently, small particles with a narrow particle size distribution are desired. Hitherto, there are, however, only limited possibilities for the preparation of monodisperse, pharmaceutically relevant dispersions. In this work, the effect of autoclaving at 121 °C on the particle size distribution of lipid nanoemulsions and -suspensions consisting of the pharmaceutically relevant components trimyristin and poloxamer 188 was studied. Additionally, the amount of emulsifier needed to stabilize both untreated and autoclaved particles was assessed. In our study, four dispersions of mean particle sizes from 45 to 150 nm were prepared by high-pressure melt homogenization. The particle size distribution before and after autoclaving was characterized using static and dynamic light scattering, differential scanning calorimetry, and transmission electron microscopy. Asymmetrical flow field-flow fractionation was used for particle size distribution analyses and for the determination of free poloxamer 188. Upon autoclaving, the mean particle size increased to up to 200 nm, but not proportionally to the initial size. At the same time, the particle size distribution width decreased remarkably. Heat treatment thus seems to be a promising approach to achieve the desired narrow particle size distribution of such dispersions. Related to the lipid content, suspension particles needed more emulsifier for stabilization than emulsion droplets, and smaller particles more than larger ones.

Mechanism and kinetics of the loss of poorly soluble drugs from liposomal carriers studied by a novel flow field-flow fractionation-based drug release-/transfer-assay.

Liposomes represent a versatile drug formulation approach e.g. for improving the water-solubility of poorly soluble drugs but also to achieve drug targeting and controlled release. For the latter applications it is essential that the drug remains associated with the liposomal carrier during transit in the vascular bed. A range of in vitro test methods has been suggested over the years for prediction of the release of drug from liposomal carriers. The majority of these fail to give a realistic prediction for poorly water-soluble drugs due to the intrinsic tendency of such compounds to remain associated with liposome bilayers even upon extensive dilution. Upon i.v. injection, in contrast, rapid drug loss often occurs due to drug transfer from the liposomal carriers to endogenous lipophilic sinks such as lipoproteins, plasma proteins or membranes of red blood cells and endothelial cells. Here we report on the application of a recently introduced in vitro predictive drug transfer assay based on incubation of the liposomal drug carrier with large multilamellar liposomes, the latter serving as a biomimetic model sink, using flow field-flow fractionation as a tool to separate the two types of liposomes. By quantifying the amount of drug remaining associated with the liposomal drug carrier as well as that transferred to the acceptor liposomes at distinct times of incubation, both the kinetics of drug transfer and release to the water phase could be established for the model drug p-THPP (5,10,15,20-tetrakis(4-hydroxyphenyl)21H,23H-porphine). p-THPP is structurally similar to temoporfin, a photosensitizer which is under clinical evaluation in a liposomal formulation. Mechanistic insights were gained by varying the donor-to-acceptor lipid mass ratio, size and lamellarity of the liposomes. Drug transfer kinetics from one liposome to another was found rate determining as compared to redistribution from the outermost to the inner concentric bilayers, such that the overall process could be adequately described by a single 1st order kinetic model. By varying the donor-to-acceptor lipid mass ratio in the range 1:1 to 1:10, a correlation was established between donor-to-acceptor-lipid mass ratio and transfer kinetics, which is regarded essential for scaling to physiological lipid mass ratios. By applying the assay to a series of structurally related model compounds of different bilayer affinity, transfer and release kinetics were established over the whole expected range of liposome bilayer associated drugs in terms of water solubility and lipophilicity. A very rapid transfer and considerable release from liposomes to the water phase was observed for the more water-soluble compounds Sudan II (clogP 5.45) and Sudan III (clogP 6.83). For the more lipophilic compounds, the rate of transfer from the donor liposomes followed the rank order Sudan IV (fastest)>Oil Red O>Sudan Black>p-THPP (slowest). For an equimolar donor-to-acceptor lipid mass ratio, half-lifes of transfer in the range of 12min (Sudan IV) up to 1.5h (p-THPP) were determined. In essence, the results presented here allow for both, mechanistic insights and predictions of drug loss from liposomal carriers upon exposure to biological sinks, which appear more realistic than the commonly employed in vitro release tests.

The use of asymmetrical flow field-flow fractionation with on-line detection in the study of drug retention within liposomal nanocarriers and drug transfer kinetics.

Due to their solubilizing capabilities, liposomes (phospholipid vesicles) are suited for designing formulations for intravenous administration of drug compounds which are poorly water-soluble. Despite the good in-vitro stability of such formulations with minimal drug leakage, upon i.v. injection there is a risk of premature drug loss due to drug transfer to plasma proteins and cell membranes. Here we report on the refinement of a recently introduced simple in vitro predictive tool by Hinna and colleagues in 2014, which brings small drug loaded (donor) liposomes in contact with large acceptor liposomes, the latter serving as a model mimicking biological sinks in the body. The donor- and acceptor-liposomes were subsequently separated using asymmetrical flow field-flow fractionation (AF4), during which the sample is exposed to a large volume of eluent which corresponds to a dilution factor of approximately 600. The model drug content in the donor- and acceptor fraction was quantified by on-line UV/VIS extinction measurements with correction for turbidity and by off-line HPLC measurements of collected fractions. The refined method allowed for (near) baseline separation of donor and acceptor vesicles as well as reliable quantification of the drug content not only of the donor- but now also of the acceptor-liposomes due to their improved size-homogeneity, colloidal stability and reduced turbidity. This improvement over the previously reported approach allowed for simultaneous quantification of both drug transfer and drug release to the aqueous phase. By sampling at specific incubation times, the release and transfer kinetics of the model compound p-THPP (5,10,15,20-tetrakis(4-hydroxyphenyl)21H,23H-porphine) was determined. p-THPP is structurally closely related to the photosensitizer temoporfin, which is in clinical use and under evaluation in liposomal formulations. The transfer of p-THPP to the acceptor vesicles followed 1st order kinetics with a half-life of approximately 300 min. As expected, equilibrium distribution between donor- and acceptor vesicles was proportional to the lipid mass ratio. An initial rapid transfer of p-THPP was found (∼ 5%) and investigated further by determining the extent of transfer between donor and acceptor during separation. The donor- and acceptor phase were found to be separated within few minutes and only minor (≤ 2%) transfer could be detected within the AF4 channel under the conditions applied for fractionation. These results demonstrates the potential of our AF4 based method as an in vitro tool to determine retention properties of lipophilic compounds within liposomal carriers in particular, but also within a variety of nano-particulate carriers provided that they exhibit a sufficient size difference compared to the applied colloidal acceptor phase.

Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters.

Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol)--in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and (31)P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70-415 nm upon repeated extrusion through 400 nm pore-filters, eventually ending with a size range from ≈30 to 85 nm upon extrusion through 30 nm pore size filters. While for small pores sizes (50 nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (∼ 300 nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.

ß-Cyclodextrin-dextran polymers for the solubilization of poorly soluble drugs.

The aim of this study was to assess the potential of novel ß-cyclodextrin (ßCD)-dextran polymers for drug delivery. The size distribution of ßCD-dextrans (for eventual parenteral administration), the influence of the dextran backbones on the stability of the ßCD/drug complex, the solubilization efficiency of poorly soluble drugs and drug release properties were investigated. Size analysis of different ßCD-dextrans was measured by size exclusion chromatography (SEC) and asymmetrical flow field-flow fractionation (AF4). Stability of drug/ßCD-dextrans was assessed by isothermal titration calorimetry (ITC) and molar enthalpies of complexation and equilibrium constants compared to some commercially available ßCD derivatives. For evaluation of the solubilization efficiency, phase-solubility diagrams were made employing hydrocortisone (HC) as a model of poorly soluble drugs, whereas reverse dialysis was used to detect potential drug supersaturation (increased molecularly dissolved drug concentration) as well as controlled release effects. Results indicate that all investigated ßCD-polymers are of appropriate sizes for parenteral administration. Thermodynamic results demonstrate that the presence of the dextran backbone structure does not affect the stability of the ßCD/drug complex, compared to native ßCD and commercially available derivatives. Solubility studies evidence higher solubilizing abilities of these new polymers in comparison to commercially available ßCDs, but no supersaturation states were induced. Moreover, drug release studies evidenced that diffusion of HC was influenced by the solubilization induced by the ßCD-derivatives.

Asymmetrical flow field-flow fractionation with on-line detection for drug transfer studies: a feasibility study.

Knowledge about drug retention within colloidal carriers is of uppermost importance particularly if drug targeting is anticipated. The aim of the present study was to evaluate asymmetrical flow field-flow fractionation (AF4) with on-line UV/VIS drug quantification for its suitability to determine both release and transfer of drug from liposomal carriers to a model acceptor phase consisting of large liposomes. The hydrophobic porphyrin 5,10,15,20-tetrakis(4-hydroxyphenyl)21H,23H-porphine (p-THPP), a fluorescent dye with an absorbance maximum in the visible range and structural similarity to the clinically used photosensitizer temoporfin, was used as a model drug, and two types of large liposomes were studied as a potential model acceptor phase. Efficiency of separation of small donor from large acceptor liposomes by AF4 was evaluated in dependence on the injected lipid mass using two different channel geometries. Drug quantification by on-line absorbance measurements was established by comprehensive evaluation of the size-dependent turbidity contribution in on-line UV/VIS detection and by comparison with off-line results obtained for the respective dye-loaded donor formulations (dissolved in methanol). Due to distinct differences in size, the acceptor liposomes (mean diameters ∼300-400 nm) could efficiently be separated from the donor liposomes (mean diameter ∼70 nm) with less than 4 % of p-THPP detected in the overlap region between both vesicle populations. Whereas p-THPP could accurately be determined in the fraction of small vesicles, on-line quantification was impaired in the fraction of the large acceptor liposomes due to the pronounced contribution of turbidity (about 80 % of total UV/VIS extinction signal). The AF4-based release/transfer approach suggested here was found repeatable and robust. The employed combination of AF4 with multi-angle laser light scattering furthermore provided detailed size information of the eluting sample and thus allowed to detect instabilities and/or interactions between the donor and acceptor liposomes. Drug quantification by on-line absorbance measurements was found feasible for the chosen model drug, but careful (re-)evaluation of turbidity effects is crucial for other drug and carrier combinations.

Influence of massage and occlusion on the ex vivo skin penetration of rigid liposomes and invasomes.

Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results confirm that massage is an important tool for increasing follicular penetration in ex vivo studies using Franz diffusion cells. Occlusion may reduce the efficacy of follicular penetration depending on the specific liposomal preparation. Rigidity in particular appears to be a relevant parameter.

Comparative SAXS and DSC study on stratum corneum structural organization in an epidermal cell culture model (ROC): impact of cultivation time.

Cell cultured skin equivalents present an alternative for dermatological in vitro evaluations of drugs and excipients as they provide the advantage of availability, lower variability and higher assay robustness compared to native skin. For penetration/permeation studies, an adequate stratum corneum barrier similar to that of human stratum corneum is, however, a prerequisite. In this study, the stratum corneum lipid organization in an epidermal cell culture model based on rat epidermal keratinocytes (REK organotypic culture, ROC) was investigated by small-angle X-ray scattering (SAXS) in dependence on ROC cultivation time and in comparison to native human and rat stratum cornea. In addition, the thermal phase behavior was studied by differential scanning calorimetry (DSC) and barrier properties were checked by measurements of the permeability of tritiated water. The development of the barrier of ROC SC obtained at different cultivation times (7, 14 and 21 days at the air-liquid interface) was connected with an increase in structural order of the SC lipids in SAXS measurements: Already cultivation for 14 days at the air-liquid interface resulted overall in a competent SC permeability barrier and SC lipid organization. Cultivation for 21 days resulted in further minor changes in the structural organization of ROC SC. The SAXS patterns of ROC SC had overall large similarities with that of human SC and point to the presence of a long periodicity phase with a repeat distance of about 122Å, e.g. slightly smaller than that determined for human SC in the present study (127Å). Moreover, SAXS results also indicate the presence of covalently bound ceramides, which are crucial for a proper SC barrier, although the corresponding thermal transitions were not clearly detectable by DSC. Due to the competent SC barrier properties and high structural and organizational similarity to that of native human SC, ROC presents a promising alternative for in vitro studies, particularly as it can be obtained under overall rather straightforward cell culture conditions and thus low assay costs.

Carbohydrate plasma expanders for passive tumor targeting: in vitro and in vivo studies.

The objective of this study was to investigate the suitability of carbohydrate plasma volume expanders as a novel polymer platform for tumor targeting. Many synthetic polymers have already been synthesized for targeted tumor therapy, but potential advantages of these carbohydrates include inexpensive synthesis, constant availability, a good safety profile, biodegradability and the long clinical use as plasma expanders. Three polymers have been tested for cytotoxicity and cytokine activation in cell cultures and conjugated with a near-infrared fluorescent dye: hydroxyethyl starches (HES 200 kDa and HES 450 kDa) and dextran (DEX 500 kDa). Particle size and molecular weight distribution were determined by asymmetric flow field-flow fractionation (AF4). The biodistribution was investigated non-invasively in nude mice using multispectral optical imaging. The most promising polymer conjugate was characterized in human colon carcinoma xenograft bearing nude mice. A tumor specific accumulation of HES 450 was observed, which proves it's potential as carrier for passive tumor targeting.

Variations in polyethylene glycol brands and their influence on the preparation process of hydrogel microspheres.

Hydrogel microspheres, e.g. for the use as protein carriers, can be prepared without the use of organic solvents via an emulsified aqueous two-phase system (ATPS) that is based on two immiscible polymer solutions. The type and concentration of the polymers can affect the ATPS and finally the distribution of incorporated drugs between the aqueous phases. For the preparation of hydrogel microspheres based on hydroxyethyl starch-hydroxyethyl methacrylate (HES-HEMA), hydroxyethyl starch-methacrylate (HES-MA), and hydroxyethyl starch-polyethylene glycol methacrylate (HES-P(EG)6MA), polyethylene glycol 12,000 (PEG 12,000) was used as second polymer. The particle size distribution and encapsulation efficiency of the microspheres depended dramatically on the type of PEG 12,000 that was used in the second phase of the ATPS. Analysis of different PEG 12,000 brands by various methods revealed differences in the salt composition and molecular weight distribution of the polymers which can explain the effects on the production process. The results illustrate that the range of product specifications may not always be tight enough to avoid variability in pharmaceutical processes like the preparation of hydrogel microspheres by an aqueous two-phase preparation process.

Analysis of liposomes using asymmetrical flow field-flow fractionation: separation conditions and drug/lipid recovery.

Liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol were analyzed by asymmetrical flow field-flow fractionation coupled with multi-angle laser light scattering. In addition to evaluation of fractionation conditions (flow conditions, sample mass, carrier liquid), radiolabeled drug-loaded liposomes were used to determine the liposome recovery and a potential loss of incorporated drug during fractionation. Neither sample concentration nor the cross-flow gradient distinctly affected the size results but at very low sample concentration (injected mass 5 µg) the fraction of larger vesicles was underestimated. Imbalance in the osmolality between the inner and outer aqueous phase resulted in liposome swelling after dilution in hypoosmotic carrier liquids. In contrast, liposome shrinking under hyperosmotic conditions was barely visible. The liposomes themselves eluted completely (lipid recoveries were close to 100%) but there was a loss of incorporated drugs during separation with a strong dependence on the octanol-water partition coefficient of the drug. Whereas corticosterone (partition coefficient ~2) was washed out more or less completely (recovery about 2%), loss of temoporfin (partition coefficient ~9) was only minor (recovery about 80%). All fractionations were well repeatable under the experimental conditions applied in the present study.

Asymmetric flow field-flow fractionation of superferrimagnetic iron oxide multicore nanoparticles.

Magnetic nanoparticles are very useful for various medical applications where each application requires particles with specific magnetic properties. In this paper we describe the modification of the magnetic properties of magnetic multicore nanoparticles (MCNPs) by size dependent fractionation. This classification was carried out by means of asymmetric flow field-flow fractionation (AF4). A clear increase of the particle size with increasing elution time was confirmed by multi-angle laser light scattering coupled to the AF4 system, dynamic light scattering and Brownian diameters determined by magnetorelaxometry. In this way 16 fractions of particles with different hydrodynamic diameters, ranging between around 100 and 500 nm, were obtained. A high reproducibility of the method was confirmed by the comparison of the mean diameters of fractions of several fractionation runs under identical conditions. The hysteresis curves were measured by vibrating sample magnetometry. Starting from a coercivity of 1.41 kA m(-1) for the original MCNPs the coercivity of the particles in the different fractions varied from 0.41 to 3.83 kA m(-1). In our paper it is shown for the first time that fractions obtained from a broad size distributed MCNP fluid classified by AF4 show a strong correlation between hydrodynamic diameter and magnetic properties. Thus we state that AF4 is a suitable technology for reproducible size dependent classification of magnetic multicore nanoparticles suspended as ferrofluids.

Liposomes as vehicles for water insoluble platinum-based potential drug: 2-(4-(tetrahydro-2H-pyran-2-yloxy)-undecyl)-propane-1,3-diamminedichloroplatinum(II).

Formulation of liposome delivery system loaded with water insoluble 2-(4-(tetrahydro-2H-pyran-2-yloxy)-undecyl)-propane-1,3-diamminedichloroplatinum(II), LipoTHP-C11 was carried out. The particle size distributions were determined by dynamic light scattering and asymmetrical flow field-flow fractionation indicating size of around 120 nm. Stability study showed that LipoTHP-C11 was stable at 4 °C for more than two months. To test suitability of chosen formulation, LipoTHP-C11 was investigated against several tumor cell lines: H12.1, 1411HP, 518A2, A549, HT-29, MCF-7 and SW1736. Furthermore, toxicity against normal fibroblasts was examined. LipoTHP-C11 may be used as an attractive candidate for further assessment in vivo as antitumor agent.